The high‐resolution structure of a UDP‐L‐rhamnose synthase from Acanthamoeba polyphagaMimivirus

Abstract For the field of virology, perhaps one of the most paradigm‐shifting events so far in the 21st century was the identification of the giant double‐stranded DNA virus that infects amoebae. Remarkably, this virus, known as Mimivirus , has a genome that encodes for nearly 1,000 proteins, some of which are involved in the biosynthesis of unusual sugars. Indeed, the virus is coated by a layer of glycosylated fibers that contain d ‐glucose, N ‐acetyl‐ d ‐glucosamine, l ‐rhamnose, and 4‐amino‐4,6‐dideoxy‐ d ‐glucose. Here we describe a combined structural and enzymological investigation of the protein encoded by the open‐reading frame L780, which corresponds to an l ‐rhamnose synthase. The structure of the L780/NADP + /UDP‐ l ‐rhamnose ternary complex was determined to 1.45 Å resolution and refined to an overall R ‐factor of 19.9%. Each subunit of the dimeric protein adopts a bilobal‐shaped appearance with the N‐terminal domain harboring the dinucleotide‐binding site and the C‐terminal domain positioning the UDP‐sugar into the active site. The overall molecular architecture of L780 places it into the short‐chain dehydrogenase/reductase superfamily. Kinetic analyses indicate that the enzyme can function on either UDP‐ and dTDP‐sugars but displays a higher catalytic efficiency with the UDP‐linked substrate. Site‐directed mutagenesis experiments suggest that both Cys 108 and Lys 175 play key roles in catalysis. This structure represents the first model of a viral UDP‐ l ‐rhamnose synthase and provides new details into these fascinating enzymes.